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Nobuyoshi Esaki - One of the best experts on this subject based on the ideXlab platform.
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substrate specificity of Fluoroacetate dehalogenase an insight from crystallographic analysis fluorescence spectroscopy and theoretical computations
Chemistry: A European Journal, 2012Co-Authors: Tomonori Nakayama, Tatsuo Kurihara, Nobuyoshi Esaki, Takashi Kamachi, Keiji Jitsumori, Rie Omi, Ken Hirotsu, Kazunari YoshizawaAbstract:The high substrate specificity of Fluoroacetate dehalogenase was explored by using crystallographic analysis, fluorescence spectroscopy, and theoretical computations. A crystal structure for the Asp104Ala mutant of the enzyme from Burkholderia sp. FA1 complexed with Fluoroacetate was determined at 1.2 A resolution. The orientation and conformation of bound Fluoroacetate is different from those in the crystal structure of the corresponding Asp110Asn mutant of the enzyme from Rhodopseudomonas palustris CGA009 reported recently (J. Am. Chem. Soc. 2011, 133, 7461). The fluorescence of the tryptophan residues of the wild-type and Trp150Phe mutant enzymes from Burkholderia sp. FA1 incubated with Fluoroacetate and chloroacetate was measured to gain information on the environment of the tryptophan residues. The environments of the tryptophan residues were found to be different between the Fluoroacetate- and chloroacetate-bound enzymes; this would come from different binding modes of these two substrates in the active site. Docking simulations and QM/MM optimizations were performed to predict favorable conformations and orientations of the substrates. The F atom of the substrate is oriented toward Arg108 in the most stable enzyme-Fluoroacetate complex. This is a stable but unreactive conformation, in which the small O-C-F angle is not suitable for the S(N)2 displacement of the F(-) ion. The cleavage of the C-F bond is initiated by the conformational change of the substrate to a near attack conformation (NAC) in the active site. The second lowest energy conformation is appropriate for NAC; the C-O distance and the O-C-F angle are reasonable for the S(N) 2 reaction. The activation energy is greatly reduced in this conformation because of three hydrogen bonds between the leaving F atom and surrounding amino acid residues. Chloroacetate cannot reach the reactive conformation, due to the longer C-Cl bond; this results in an increase of the activation energy despite the weaker C-Cl bond.
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The Catalytic Mechanism of Fluoroacetate Dehalogenase: A Computational Exploration of Biological Dehalogenation
Chemistry - A European Journal, 2009Co-Authors: Takashi Kamachi, Tatsuo Kurihara, Nobuyoshi Esaki, Tomonori Nakayama, Osamu Shitamichi, Keiji Jitsumori, Kazunari YoshizawaAbstract:The biological dehalogenation of Fluoroacetate carried out by Fluoroacetate dehalogenase is discussed by using quantum mechanical/molecular mechanical (QM/MM) calculations for a whole-enzyme model of 10 800 atoms. Substrate Fluoroacetate is anchored by a hydrogen-bonding network with water molecules and the surrounding amino acid residues of Arg105, Arg108, His149, Trp150, and Tyr212 in the active site in a similar way to haloalkane dehalogenase. Asp104 is likely to act as a nucleophile to attack the alpha-carbon of Fluoroacetate, resulting in the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule to the carbonyl carbon atom. The cleavage of the strong C-F bond is greatly facilitated by the hydrogen-bonding interactions between the leaving fluorine atom and the three amino acid residues of His149, Trp150, and Tyr212. The hydrolysis of the ester intermediate is initiated by a proton transfer from the water molecule to His271 and by the simultaneous nucleophilic attack of the water molecule. The transition state and produced tetrahedral intermediate are stabilized by Asp128 and the oxyanion hole composed of Phe34 and Arg105.
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x ray crystallographic and mutational studies of Fluoroacetate dehalogenase from burkholderia sp strain fa1
Journal of Bacteriology, 2009Co-Authors: Keiji Jitsumori, Tatsuo Kurihara, Rie Omi, Atsushi Kurata, Hisaaki Mihara, Ikuko Miyahara, Ken Hirotsu, Nobuyoshi EsakiAbstract:Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of Fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of a carbon-fluorine bond of an aliphatic compound: the bond energy of the carbon-fluorine bond is among the highest found in natural products. The enzyme also acts on chloroacetate, although much less efficiently. We here determined the X-ray crystal structure of the enzyme from Burkholderia sp. strain FA1 as the first experimentally determined three-dimensional structure of Fluoroacetate dehalogenase. The enzyme belongs to the alpha/beta hydrolase superfamily and exists as a homodimer. Each subunit consists of core and cap domains. The catalytic triad, Asp104-His271-Asp128, of which Asp104 serves as the catalytic nucleophile, was found in the core domain at the domain interface. The active site was composed of Phe34, Asp104, Arg105, Arg108, Asp128, His271, and Phe272 of the core domain and Tyr147, His149, Trp150, and Tyr212 of the cap domain. An electron density peak corresponding to a chloride ion was found in the vicinity of the N(epsilon1) atom of Trp150 and the N(epsilon2) atom of His149, suggesting that these are the halide ion acceptors. Site-directed replacement of each of the active-site residues, except for Trp150, by Ala caused the total loss of the activity toward Fluoroacetate and chloroacetate, whereas the replacement of Trp150 caused the loss of the activity only toward Fluoroacetate. An interaction between Trp150 and the fluorine atom is probably an absolute requirement for the reduction of the activation energy for the cleavage of the carbon-fluorine bond.
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reactivity of asparagine residue at the active site of the d105n mutant of Fluoroacetate dehalogenase from moraxella sp b
Biochimica et Biophysica Acta, 2004Co-Authors: Susumu Ichiyama, Tatsuo Kurihara, Susumu Tsunasawa, Haruhiko Kawasaki, Yoshifumi Kogure, Nobuyoshi EsakiAbstract:Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes cleavage of the carbon-fluorine bond of Fluoroacetate, whose dissociation energy is among the highest found in natural products. Asp105 functions as the catalytic nucleophile that attacks the alpha-carbon atom of the substrate to displace the fluorine atom. In spite of the essential role of Asp105, we found that site-directed mutagenesis to replace Asp105 by Asn does not result in total inactivation of the enzyme. The activity of the mutant enzyme increased in a time- and temperature-dependent manner. We analyzed the enzyme by ion-spray mass spectrometry and found that the reactivation was caused by the hydrolytic deamidation of Asn105 to generate the wild-type enzyme. Unlike Asn10 of the l-2-haloacid dehalogenase (L-DEX YL) D10N mutant, Asn105 of the Fluoroacetate dehalogenase D105N mutant did not function as a nucleophile to catalyze the dehalogenation.
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Purification, characterization, and gene cloning of a novel Fluoroacetate dehalogenase from Burkholderia sp. FA1
Journal of Molecular Catalysis B-enzymatic, 2003Co-Authors: Tatsuo Kurihara, Takahiro Yamauchi, Susumu Ichiyama, Hiroyuki Takahata, Nobuyoshi EsakiAbstract:Abstract Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of Fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of the highly stable carbon–fluorine bond in an aliphatic compound. The bacterial isolate FA1, which was identified as Burkholderia, grew on Fluoroacetate as the sole carbon source to produce Fluoroacetate dehalogenase (FAc-DEX FA1). The enzyme was purified to homogeneity and characterized. The molecular weights were estimated to be 79,000 and 34,000 by gel filtration and SDS-polyacrylamide gel electrophoresis (PAGE), respectively, suggesting that the enzyme is a dimer. The purified enzyme was specific to haloacetates, and Fluoroacetate was the best substrate. The activities toward chloroacetate and bromoacetate were less than 5% of the activity toward Fluoroacetate. The Km and Vmax values for the hydrolysis of Fluoroacetate were 5.1 mM and 11 μmol per minute milligram, respectively. The gene coding for the enzyme was isolated, and the nucleotide sequence was determined. The open reading frame consisted of 912 nucleotides, corresponding to 304 amino acid residues. Although FAc-DEX FA1 showed high sequence similarity to Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX H1) (61% identity), the substrate specificity of FAc-DEX FA1 was significantly different from that of FAc-DEX H1: FAc-DEX FA1 was more specific to Fluoroacetate than FAc-DEX H1.
Tatsuo Kurihara - One of the best experts on this subject based on the ideXlab platform.
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substrate specificity of Fluoroacetate dehalogenase an insight from crystallographic analysis fluorescence spectroscopy and theoretical computations
Chemistry: A European Journal, 2012Co-Authors: Tomonori Nakayama, Tatsuo Kurihara, Nobuyoshi Esaki, Takashi Kamachi, Keiji Jitsumori, Rie Omi, Ken Hirotsu, Kazunari YoshizawaAbstract:The high substrate specificity of Fluoroacetate dehalogenase was explored by using crystallographic analysis, fluorescence spectroscopy, and theoretical computations. A crystal structure for the Asp104Ala mutant of the enzyme from Burkholderia sp. FA1 complexed with Fluoroacetate was determined at 1.2 A resolution. The orientation and conformation of bound Fluoroacetate is different from those in the crystal structure of the corresponding Asp110Asn mutant of the enzyme from Rhodopseudomonas palustris CGA009 reported recently (J. Am. Chem. Soc. 2011, 133, 7461). The fluorescence of the tryptophan residues of the wild-type and Trp150Phe mutant enzymes from Burkholderia sp. FA1 incubated with Fluoroacetate and chloroacetate was measured to gain information on the environment of the tryptophan residues. The environments of the tryptophan residues were found to be different between the Fluoroacetate- and chloroacetate-bound enzymes; this would come from different binding modes of these two substrates in the active site. Docking simulations and QM/MM optimizations were performed to predict favorable conformations and orientations of the substrates. The F atom of the substrate is oriented toward Arg108 in the most stable enzyme-Fluoroacetate complex. This is a stable but unreactive conformation, in which the small O-C-F angle is not suitable for the S(N)2 displacement of the F(-) ion. The cleavage of the C-F bond is initiated by the conformational change of the substrate to a near attack conformation (NAC) in the active site. The second lowest energy conformation is appropriate for NAC; the C-O distance and the O-C-F angle are reasonable for the S(N) 2 reaction. The activation energy is greatly reduced in this conformation because of three hydrogen bonds between the leaving F atom and surrounding amino acid residues. Chloroacetate cannot reach the reactive conformation, due to the longer C-Cl bond; this results in an increase of the activation energy despite the weaker C-Cl bond.
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The Catalytic Mechanism of Fluoroacetate Dehalogenase: A Computational Exploration of Biological Dehalogenation
Chemistry - A European Journal, 2009Co-Authors: Takashi Kamachi, Tatsuo Kurihara, Nobuyoshi Esaki, Tomonori Nakayama, Osamu Shitamichi, Keiji Jitsumori, Kazunari YoshizawaAbstract:The biological dehalogenation of Fluoroacetate carried out by Fluoroacetate dehalogenase is discussed by using quantum mechanical/molecular mechanical (QM/MM) calculations for a whole-enzyme model of 10 800 atoms. Substrate Fluoroacetate is anchored by a hydrogen-bonding network with water molecules and the surrounding amino acid residues of Arg105, Arg108, His149, Trp150, and Tyr212 in the active site in a similar way to haloalkane dehalogenase. Asp104 is likely to act as a nucleophile to attack the alpha-carbon of Fluoroacetate, resulting in the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule to the carbonyl carbon atom. The cleavage of the strong C-F bond is greatly facilitated by the hydrogen-bonding interactions between the leaving fluorine atom and the three amino acid residues of His149, Trp150, and Tyr212. The hydrolysis of the ester intermediate is initiated by a proton transfer from the water molecule to His271 and by the simultaneous nucleophilic attack of the water molecule. The transition state and produced tetrahedral intermediate are stabilized by Asp128 and the oxyanion hole composed of Phe34 and Arg105.
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x ray crystallographic and mutational studies of Fluoroacetate dehalogenase from burkholderia sp strain fa1
Journal of Bacteriology, 2009Co-Authors: Keiji Jitsumori, Tatsuo Kurihara, Rie Omi, Atsushi Kurata, Hisaaki Mihara, Ikuko Miyahara, Ken Hirotsu, Nobuyoshi EsakiAbstract:Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of Fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of a carbon-fluorine bond of an aliphatic compound: the bond energy of the carbon-fluorine bond is among the highest found in natural products. The enzyme also acts on chloroacetate, although much less efficiently. We here determined the X-ray crystal structure of the enzyme from Burkholderia sp. strain FA1 as the first experimentally determined three-dimensional structure of Fluoroacetate dehalogenase. The enzyme belongs to the alpha/beta hydrolase superfamily and exists as a homodimer. Each subunit consists of core and cap domains. The catalytic triad, Asp104-His271-Asp128, of which Asp104 serves as the catalytic nucleophile, was found in the core domain at the domain interface. The active site was composed of Phe34, Asp104, Arg105, Arg108, Asp128, His271, and Phe272 of the core domain and Tyr147, His149, Trp150, and Tyr212 of the cap domain. An electron density peak corresponding to a chloride ion was found in the vicinity of the N(epsilon1) atom of Trp150 and the N(epsilon2) atom of His149, suggesting that these are the halide ion acceptors. Site-directed replacement of each of the active-site residues, except for Trp150, by Ala caused the total loss of the activity toward Fluoroacetate and chloroacetate, whereas the replacement of Trp150 caused the loss of the activity only toward Fluoroacetate. An interaction between Trp150 and the fluorine atom is probably an absolute requirement for the reduction of the activation energy for the cleavage of the carbon-fluorine bond.
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reactivity of asparagine residue at the active site of the d105n mutant of Fluoroacetate dehalogenase from moraxella sp b
Biochimica et Biophysica Acta, 2004Co-Authors: Susumu Ichiyama, Tatsuo Kurihara, Susumu Tsunasawa, Haruhiko Kawasaki, Yoshifumi Kogure, Nobuyoshi EsakiAbstract:Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes cleavage of the carbon-fluorine bond of Fluoroacetate, whose dissociation energy is among the highest found in natural products. Asp105 functions as the catalytic nucleophile that attacks the alpha-carbon atom of the substrate to displace the fluorine atom. In spite of the essential role of Asp105, we found that site-directed mutagenesis to replace Asp105 by Asn does not result in total inactivation of the enzyme. The activity of the mutant enzyme increased in a time- and temperature-dependent manner. We analyzed the enzyme by ion-spray mass spectrometry and found that the reactivation was caused by the hydrolytic deamidation of Asn105 to generate the wild-type enzyme. Unlike Asn10 of the l-2-haloacid dehalogenase (L-DEX YL) D10N mutant, Asn105 of the Fluoroacetate dehalogenase D105N mutant did not function as a nucleophile to catalyze the dehalogenation.
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Purification, characterization, and gene cloning of a novel Fluoroacetate dehalogenase from Burkholderia sp. FA1
Journal of Molecular Catalysis B-enzymatic, 2003Co-Authors: Tatsuo Kurihara, Takahiro Yamauchi, Susumu Ichiyama, Hiroyuki Takahata, Nobuyoshi EsakiAbstract:Abstract Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of Fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of the highly stable carbon–fluorine bond in an aliphatic compound. The bacterial isolate FA1, which was identified as Burkholderia, grew on Fluoroacetate as the sole carbon source to produce Fluoroacetate dehalogenase (FAc-DEX FA1). The enzyme was purified to homogeneity and characterized. The molecular weights were estimated to be 79,000 and 34,000 by gel filtration and SDS-polyacrylamide gel electrophoresis (PAGE), respectively, suggesting that the enzyme is a dimer. The purified enzyme was specific to haloacetates, and Fluoroacetate was the best substrate. The activities toward chloroacetate and bromoacetate were less than 5% of the activity toward Fluoroacetate. The Km and Vmax values for the hydrolysis of Fluoroacetate were 5.1 mM and 11 μmol per minute milligram, respectively. The gene coding for the enzyme was isolated, and the nucleotide sequence was determined. The open reading frame consisted of 912 nucleotides, corresponding to 304 amino acid residues. Although FAc-DEX FA1 showed high sequence similarity to Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX H1) (61% identity), the substrate specificity of FAc-DEX FA1 was significantly different from that of FAc-DEX H1: FAc-DEX FA1 was more specific to Fluoroacetate than FAc-DEX H1.
U Sonnewald - One of the best experts on this subject based on the ideXlab platform.
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trafficking of amino acids between neurons and glia in vivo effects of inhibition of glial metabolism by Fluoroacetate
Journal of Cerebral Blood Flow and Metabolism, 1997Co-Authors: Bjornar Hassel, H S Bachelard, Paula Jones, Frode Fonnum, U SonnewaldAbstract:Glial-neuronal interchange of amino acids was studied by 13C nuclear magnetic resonance spectroscopy of brain extracts from Fluoroacetate-treated mice that received [1,2-(13)C]acetate and [1-(13)C]glucose simultaneously. [13C]Acetate was found to be a specific marker for glial metabolism even with the large doses necessary for nuclear magnetic resonance spectroscopy. Fluoroacetate, 100 mg/kg, blocked the glial, but not the neuronal tricarboxylic acid cycles as seen from the 13C labeling of glutamine, glutamate, and gamma-aminobutyric acid. Glutamine, but not citrate, was the only glial metabolite that could account for the transfer of 13C from glia to neurons. Massive glial uptake of transmitter glutamate was indicated by the labeling of glutamine from [1-(13)C]glucose in Fluoroacetate-treated mice. The C-3/C-4 enrichment ratio, which indicates the degree of cycling of label, was higher in glutamine than in glutamate in the presence of Fluoroacetate, suggesting that transmitter glutamate (which was converted to glutamine after release) is associated with a tricarboxylic acid cycle that turns more rapidly than the overall cerebral tricarboxylic acid cycle.
Cormac D. Murphy - One of the best experts on this subject based on the ideXlab platform.
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Comparison of planktonic and biofilm cultures of Pseudomonas fluorescens DSM 8341 cells grown on Fluoroacetate.
Applied and Environmental Microbiology, 2009Co-Authors: Barry Heffernan, Cormac D. Murphy, Eoin CaseyAbstract:Comparisons between the physiological properties of Pseudomonas fluorescens biofilm cells grown in a tubular biofilm reactor and planktonic cells grown in a chemostat were performed. Fluoroacetate was the sole carbon source for all experiments. The performance of cells was assessed using cell cycle kinetics and by determining specific Fluoroacetate utilization rates. Cell cycle kinetics were studied by flow cytometry in conjunction with the fluorescent stain propidium iodide. Determination of the DNA content of planktonic and biofilm cultures showed little difference between the two modes of growth. Cultures with comparable specific glycolate utilization rates had similar percentages of cells in the B phase of the cell cycle, indicating similar growth rates. Specific Fluoroacetate utilization rates showed the performance of planktonic cells to be superior to that of biofilm cells, with more Fluoroacetate utilized per cell at similar specific Fluoroacetate loading rates. A consequence of this decreased biofilm performance was the accumulation of glycolate in the effluent of biofilm cultures. This accumulation of glycolate was not observed in the effluent of planktonic cultures. Spatial stratification of oxygen within the biofilm was identified as a possible explanation for the overflow metabolism of glycolate and the decreased performance of the biofilm cells.
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Treatment of Fluoroacetate by a Pseudomonas fluorescens Biofilm Grown in Membrane Aerated Biofilm Reactor
Environmental Science & Technology, 2009Co-Authors: Barry Heffernan, Cormac D. Murphy, Eoin Syron, Eoin CaseyAbstract:Fluorinated organic compounds have widespread applications, and their accumulation in the environment is a concern. Biofilm reactors are an effective technology for the treatment of contaminated wastewater, yet almost no research has been conducted on the effectiveness of biofilms for the biodegradation of fluorinated aliphatic compounds. In this paper we describe experiments undertaken to investigate the degradation of Fluoroacetate using a membrane aerated biofilm reactor (MABR) by Pseudomonas fluorescens DSM8341. The concentration of Fluoroacetate in the medium influenced biofilm structure, with less dense biofilm observed at lower Fluoroacetate loading rates. As biofilm thickness increased, oxygen utilization decreased, probably as a consequence of increased resistance to oxygen transfer. Furthermore, most of the biofilm was anaerobic, since oxygen penetration depth was less than 1000 μm. Biofilm performance, in terms of Fluoroacetate removal efficiency, was improved by decreasing the Fluoroacetate lo...
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Purification and properties of Fluoroacetate dehalogenase from Pseudomonas fluorescens DSM 8341
Biotechnology Letters, 2008Co-Authors: Clár Donnelly, Cormac D. MurphyAbstract:The degradation of Fluoroacetate by microorganisms has been established for some time, although only a handful of dehalogenases capable of hydrolyzing the stable C-F bond have been studied. Pseudomonas fluorescens DSM 8341 was originally isolated from soil and readily degrades Fluoroacetate, thus it was thought that its dehalogenase might have some desirable properties. The enzyme was purified from cell-free extracts and characterised: it is a monomer of 32,500 Da, with a pH optimum of 8 and is stable between pH 4 and 10; its activity is stimulated by some metal ions (Mg(2+), Mn(2+) and Fe(3+)), but inhibited by others (Hg(2+), Ag(2+)). The enzyme is specific for Fluoroacetate, and the K(m) for this substrate (0.68 mM) is the lowest determined for enzymes of this type that have been investigated to date.
Rie Omi - One of the best experts on this subject based on the ideXlab platform.
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substrate specificity of Fluoroacetate dehalogenase an insight from crystallographic analysis fluorescence spectroscopy and theoretical computations
Chemistry: A European Journal, 2012Co-Authors: Tomonori Nakayama, Tatsuo Kurihara, Nobuyoshi Esaki, Takashi Kamachi, Keiji Jitsumori, Rie Omi, Ken Hirotsu, Kazunari YoshizawaAbstract:The high substrate specificity of Fluoroacetate dehalogenase was explored by using crystallographic analysis, fluorescence spectroscopy, and theoretical computations. A crystal structure for the Asp104Ala mutant of the enzyme from Burkholderia sp. FA1 complexed with Fluoroacetate was determined at 1.2 A resolution. The orientation and conformation of bound Fluoroacetate is different from those in the crystal structure of the corresponding Asp110Asn mutant of the enzyme from Rhodopseudomonas palustris CGA009 reported recently (J. Am. Chem. Soc. 2011, 133, 7461). The fluorescence of the tryptophan residues of the wild-type and Trp150Phe mutant enzymes from Burkholderia sp. FA1 incubated with Fluoroacetate and chloroacetate was measured to gain information on the environment of the tryptophan residues. The environments of the tryptophan residues were found to be different between the Fluoroacetate- and chloroacetate-bound enzymes; this would come from different binding modes of these two substrates in the active site. Docking simulations and QM/MM optimizations were performed to predict favorable conformations and orientations of the substrates. The F atom of the substrate is oriented toward Arg108 in the most stable enzyme-Fluoroacetate complex. This is a stable but unreactive conformation, in which the small O-C-F angle is not suitable for the S(N)2 displacement of the F(-) ion. The cleavage of the C-F bond is initiated by the conformational change of the substrate to a near attack conformation (NAC) in the active site. The second lowest energy conformation is appropriate for NAC; the C-O distance and the O-C-F angle are reasonable for the S(N) 2 reaction. The activation energy is greatly reduced in this conformation because of three hydrogen bonds between the leaving F atom and surrounding amino acid residues. Chloroacetate cannot reach the reactive conformation, due to the longer C-Cl bond; this results in an increase of the activation energy despite the weaker C-Cl bond.
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x ray crystallographic and mutational studies of Fluoroacetate dehalogenase from burkholderia sp strain fa1
Journal of Bacteriology, 2009Co-Authors: Keiji Jitsumori, Tatsuo Kurihara, Rie Omi, Atsushi Kurata, Hisaaki Mihara, Ikuko Miyahara, Ken Hirotsu, Nobuyoshi EsakiAbstract:Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of Fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of a carbon-fluorine bond of an aliphatic compound: the bond energy of the carbon-fluorine bond is among the highest found in natural products. The enzyme also acts on chloroacetate, although much less efficiently. We here determined the X-ray crystal structure of the enzyme from Burkholderia sp. strain FA1 as the first experimentally determined three-dimensional structure of Fluoroacetate dehalogenase. The enzyme belongs to the alpha/beta hydrolase superfamily and exists as a homodimer. Each subunit consists of core and cap domains. The catalytic triad, Asp104-His271-Asp128, of which Asp104 serves as the catalytic nucleophile, was found in the core domain at the domain interface. The active site was composed of Phe34, Asp104, Arg105, Arg108, Asp128, His271, and Phe272 of the core domain and Tyr147, His149, Trp150, and Tyr212 of the cap domain. An electron density peak corresponding to a chloride ion was found in the vicinity of the N(epsilon1) atom of Trp150 and the N(epsilon2) atom of His149, suggesting that these are the halide ion acceptors. Site-directed replacement of each of the active-site residues, except for Trp150, by Ala caused the total loss of the activity toward Fluoroacetate and chloroacetate, whereas the replacement of Trp150 caused the loss of the activity only toward Fluoroacetate. An interaction between Trp150 and the fluorine atom is probably an absolute requirement for the reduction of the activation energy for the cleavage of the carbon-fluorine bond.